WO2021065766A1 - Procédé de criblage de cellules produisant des sécrétions et kit de criblage de cellules produisant des sécrétions - Google Patents

Procédé de criblage de cellules produisant des sécrétions et kit de criblage de cellules produisant des sécrétions Download PDF

Info

Publication number
WO2021065766A1
WO2021065766A1 PCT/JP2020/036534 JP2020036534W WO2021065766A1 WO 2021065766 A1 WO2021065766 A1 WO 2021065766A1 JP 2020036534 W JP2020036534 W JP 2020036534W WO 2021065766 A1 WO2021065766 A1 WO 2021065766A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
secretion
cell
screening
well
Prior art date
Application number
PCT/JP2020/036534
Other languages
English (en)
Japanese (ja)
Inventor
享史 大坂
Original Assignee
東京応化工業株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 東京応化工業株式会社 filed Critical 東京応化工業株式会社
Priority to EP20871602.7A priority Critical patent/EP4039789A4/fr
Priority to US17/754,049 priority patent/US20220381768A1/en
Priority to JP2021551221A priority patent/JPWO2021065766A1/ja
Priority to KR1020227010340A priority patent/KR20220070220A/ko
Priority to CN202080067740.0A priority patent/CN114450594A/zh
Publication of WO2021065766A1 publication Critical patent/WO2021065766A1/fr

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/06—Methods of screening libraries by measuring effects on living organisms, tissues or cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B01L3/50255—Multi-well filtration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5038—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving detection of metabolites per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a method for screening secretion-producing cells and a screening kit for secretion-producing cells.
  • the present application claims priority based on Japanese Patent Application No. 2019-178557 filed in Japan on September 30, 2019, the contents of which are incorporated herein by reference.
  • the target of cell analysis has been subdivided from the cell group level to the single cell level. Attempts have been made to capture, identify, sort, collect, culture, perform genetic analysis, and use the sorted cells at the individual level.
  • a method for identifying and selecting cells a method is adopted in which the secretion secreted from the cells is separated, the target secretion is detected, and then the cells secreting the target secretion are screened.
  • a technique for collectively analyzing a plurality of cells is useful.
  • Techniques for batch analysis of cells include, for example, spot-incubating a mixture of antibody-producing cells, antigen-binding beads and fluorescently labeled anti-antibody-antibodies on a glass slide to localize the fluorescence surrounding the antibody-producing cells.
  • a method for identifying B cells producing the target antibody by the increase has been reported (Patent Document 1).
  • the present invention has been made in view of the above circumstances, and is a method for screening secretory-producing cells and a screening kit for the secretory-producing cells, which can accurately obtain cells producing the target secretion.
  • the challenge is to provide.
  • the present invention has adopted the following configuration.
  • a first aspect of the present invention is a method for screening a secretion-producing cell that screens a cell that produces a target secretion from a plurality of cells, and has a plurality of through-holes at the bottom having a size that the cell does not pass through.
  • a step A in which the cells and detection particles capable of capturing the target secretion and having a size that does not pass through the through holes are captured in the wells, and captured in the plurality of wells.
  • a step D for identifying a well in which a producing cell is captured is included, and the well has a size capable of capturing the cell in a cell-by-cell unit while capturing one or more of the detected particles. This is a method for screening secretion-producing cells.
  • a second aspect of the present invention is a secretion-producing cell screening kit for screening cells that produce a desired secretion from a plurality of cells, comprising a device containing a plurality of wells, carrier particles, and the like.
  • the well has a through hole at the bottom having a size capable of capturing the cells on a cell-by-cell basis in a state of capturing one or more of the carrier particles, and having a size through which the cells do not pass.
  • the carrier particles are a screening kit for secretory-producing cells having a size that does not pass through the through-holes.
  • the present invention it is possible to provide a method for screening secretion-producing cells and a screening kit for the secretion-producing cells, which can accurately obtain cells producing the target secretion.
  • step A of the screening method of a secretion-producing cell It is a figure explaining an example of step B of the screening method of a secretion-producing cell. It is a figure explaining an example of step C of the screening method of a secretion-producing cell. It is a figure explaining an example of step D of the screening method of a secretion-producing cell. It is a figure explaining the modification of the step A of the screening method of a secretion-producing cell. It is a figure explaining an example of the screening method of the secretion-producing cell when the detection particle is a cell. It is a figure explaining an example of the washing process of the screening method of a secretion-producing cell.
  • FIG. 3 is a fluorescence micrograph of a well of an example to which the screening method according to the embodiment is applied.
  • A is a fluorescence micrograph of a well that has not been washed.
  • B is a fluorescence micrograph of the wells subjected to the washing step.
  • a first aspect of the present invention is a secretory-producing cell screening method for screening cells that produce a target secretion from a plurality of cells.
  • the detection particles capable of capturing the cells and the target secretion in a plurality of wells having through holes of a size that the cells do not pass through are large enough not to pass through the through holes.
  • a step D of identifying a well in which cells producing the target secretion from the plurality of wells are captured is included.
  • the well has a size capable of capturing the cells on a cell-by-cell basis in a state where one or more of the detected particles are captured.
  • the cell C and the detection particle B capable of capturing the target secretion are captured in a plurality of wells 50 having a through hole 52 at the bottom having a size that the cell C to be screened does not pass through. And are captured (step A; FIG. 1).
  • the detection particle B has a size that does not pass through the through hole 52.
  • the well 50 has a size capable of capturing cells C on a cell-by-cell basis in a state where one or more detection particles B are captured. Cell C captured in well 50 is then incubated to cause cell C to produce secretion A (step B; FIG. 2).
  • step C the target secretion A captured by the detection particles B is detected using the detection reagent D or the like (step C; FIG. 3).
  • step C the target secretion A (secretion A-1) is produced from a plurality of wells 50 (wells 50-1, 50-2, 50-3).
  • the well 50 (well 50-1) from which the cells were captured is identified (step D; FIG. 4).
  • the plurality of cells used in the screening method according to the present embodiment are a cell population predicted to contain a target secretion-producing cell.
  • the plurality of cells is preferably a population of cells that produce secretions. Examples of cells include mammal (human, mouse, rat, rabbit, horse, camel, monkey, etc.) cells, birds (chicken, etc.) cells, insect (kai, etc.) cells and other animal cells; plant cells; fungi such as yeast. ; Bacteria such as Escherichia coli and the like can be mentioned.
  • the screening cell may be a recombinant cell in which a gene encoding a secretory protein or a secretory peptide is introduced into these cells.
  • the secretory protein and secretory peptide may be a non-secretory protein or a chimeric protein in which a secretory signal peptide is linked to a non-secretory protein or a non-secretory peptide.
  • the target secretion may be of natural origin or may be non-natural using genetic engineering.
  • the target secretion is preferably a single secretion, but not limited to.
  • examples of the target secretion include immunoglobulins (immunoglobulin G (IgG), immunoglobulin M (IgM), etc.); interleukins (IL-2, IL-7, IL-12, IL-15, etc.), chemokine, and the like.
  • Interferon IFN- ⁇ , etc.
  • hematopoietic factors colony stimulator, granulocyte colony stimulator, erythropoetin, etc.
  • cell growth factors epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, hepatocellular growth factor, trans
  • Cythins such as forming growth factors
  • cytotoxic factors tumor necrosis factors, lymphoxins
  • adipokines lactin secreted from adipose tissue, tumor necrosis factors, etc.
  • neurotrophic factors neurotrophic factors (nerve growth factors, etc.); antibiotics ; Metabolic products of microorganisms such as pigments; Hormones (peptide hormones, steroid hormones, microbial hormones, etc.); Chimera proteins of secretory signal peptides and non-secretory proteins, etc., but are not limited thereto.
  • the target secretion is not limited to a natural protein or a natural peptide, and
  • the secretion is preferably an antibody.
  • the natural antibody include immunoglobulin G (IgG), immunoglobulin M (IgM) and the like.
  • the unnatural antibody include antibody fragments such as Fab, scFv, and Diabody; single domain antibodies (Single Domain Antibodies, Methods in Molecular Biology Volume, 911, 2012); artificial protein molecules having antibody-like properties (S Strukelj B, Berlek A. Non-immunoglobulin scaffolds: a focal antibody, Trends Biotechnol., 2015, Apr 27) and the like.
  • secretion-producing cells that produce the target secretion include antibody-producing cells, cytokine-producing cells, and hormone-secreting cells.
  • antibody molecule-producing cells include, but are not limited to, B cells, hybridomas in which B cells and myeloma cells are fused, and recombinant cells in which a polynucleotide encoding an antibody molecule is introduced into the cells.
  • cells into which an antibody gene is introduced include animal cells, fungi such as yeast, and bacteria such as Escherichia coli.
  • NSO cells, CHO cells, COS cells, 293FT cells and the like can be used as the cells into which the antibody gene is introduced.
  • cytokine-producing cells include, but are not limited to, macrophages, B cells, T cells, NK cells, NKT cells, dendritic cells, hepatic Kupffer cells, stromal cells, fibroblasts, and vascular endothelial cells.
  • Hormone-secreting cells include, but are not limited to, anterior lobe cells, somatotropin-producing cells, lactotropin-producing cells, thyroid stimulating hormone-producing cells, gonad-stimulating hormone-producing cells, corticotropin-producing cells, intermediate pituitary cells, and melanin cell stimulation.
  • Hormone-secreting cells oxytocin-secreting cells, vasopressin-secreting cells, serotonin-secreting cells, endolphin-secreting cells, somatostatin-secreting cells, gastrin-secreting cells, secretin-secreting cells, cholecystkinin-secreting cells, insulin-secreting cells, glucagon-secreting cells, bombesin-secreting Cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid main cells, anoxic cells, adrenal gland cells, chromium-affinitive cells, steroid hormone (mineral corticoid or sugar corticoid) -producing cells, testosterone Examples thereof include secretory cells, estrogen-secreting cells, progesterone-secreting cells, kidney parafilamental apparatus cells, kidney compact spot cells, kidney perivascular pole cells, and kidney mesangium cells.
  • the target secretion is preferably an antibody
  • the secretion-producing cell is preferably an antibody-producing cell.
  • Step A a plurality of wells having through-holes having a size that cells do not pass through are provided with detection particles that can capture the cells and the target secretion and have a size that does not pass through the through-holes. The detection particles and the detection particles are captured.
  • FIG. 1 is a diagram illustrating an example of step A.
  • the device 100 includes a plurality of wells 50.
  • the well 50 has a through hole 52 in the bottom portion 50a.
  • the device 100 may be composed of, for example, a substrate having a first surface and a second surface, a membrane, or the like.
  • the plurality of wells 50 may be recesses having an opening on the first surface of the substrate or membrane, for example.
  • the through hole 52 may be a through hole that penetrates from the first surface to the second surface of the substrate or membrane.
  • the material of the device 100 is preferably not harmful to cells.
  • Examples of the material of the device 100 include glass; general resins such as polyethylene terephthalate (PET), polymethylmethacrylate (PMMA), polycarbonate (PC), polystyrene (PS), cycloolefin polymer (COP), and epoxy. Can be mentioned.
  • general resins such as polyethylene terephthalate (PET), polymethylmethacrylate (PMMA), polycarbonate (PC), polystyrene (PS), cycloolefin polymer (COP), and epoxy.
  • PET polyethylene terephthalate
  • PMMA polymethylmethacrylate
  • PC polycarbonate
  • PS polystyrene
  • COP cycloolefin polymer
  • epoxy cycloolefin polymer
  • the planar shape of the opening of the well 50 is not particularly limited as long as it can capture the screening cells C on a cell-by-cell basis.
  • Examples of the planar shape of the well 50 include a circle, an ellipse, a polygon (a quadrangle such as a square, a hexagon, an octagon, etc.). From the viewpoint of increasing the arrangement density, a regular hexagon is preferable.
  • the bottom 50a of the well 50 may have a flat bottom or a round bottom. A flat bottom is preferable from the viewpoint of the ease of forming the through hole 52 and the viewpoint that the amount of the detected particles B stored can be increased.
  • the well 50 has a size capable of capturing screening cells C on a cell-by-cell basis in a state where one or more detection particles B, which will be described later, are captured.
  • the single cell unit means, for example, a single cell.
  • the size of the well 50 can be appropriately selected according to the size of the screening cell C.
  • the size of the well 50 is not limited, but generally, the maximum diameter of the circle entering the well opening can be about 1 to 100 ⁇ m, and the depth can be about 1 to 100 ⁇ m.
  • the maximum diameter of the circle entering the well opening is more preferably 2 to 50 ⁇ m, even more preferably 3 to 25 ⁇ m.
  • the depth of the well 50 is more preferably 2 to 70 ⁇ m, even more preferably 3 to 50 ⁇ m, and particularly preferably 4 to 30 ⁇ m.
  • the maximum diameter of the circle entering the well 50 in a plan view can be about 0.5 to 2 times the maximum diameter of the screening cell C, which is preferable. Is 0.8 to 1.9 times.
  • the depth of the well 50 can be about 0.5 to 4 times the maximum diameter of the screening cell C, and more preferably 0.8 to 1.9 times.
  • the through hole 52 is provided in the bottom portion 50a of the well 50 and communicates the internal space of the well 50 with the external space.
  • the through hole 52 is a through hole that penetrates the member forming the bottom portion 50a.
  • the position, shape, size, etc. of the through hole 52 are not particularly limited as long as the screening cell C does not pass through and the liquid containing the secretion secreted by the screening cell C can pass through.
  • Examples of the planar shape of the through hole 52 include a circle, an ellipse, a polygon (a quadrangle such as a square, a hexagon, an octagon, etc.). From the viewpoint of ease of formation, a circular shape is preferable.
  • the size of the through hole 52 is not limited, but generally, the minimum inner diameter of the through hole 52 can be 10 nm to 20 ⁇ m.
  • the minimum inner diameter of the through hole 52 is preferably 50 nm to 15 ⁇ m, more preferably 100 nm to 10 ⁇ m.
  • the minimum inner diameter of the through hole 52 is preferably 0.5 times or less, more preferably 0.1 times or less the maximum diameter of the screening cell C, and 0. It is more preferably 05 times or less.
  • the detection particle B is a particle capable of capturing the target secretion.
  • the detection particle B has a size that is captured by the well 50 and does not pass through the through hole 52.
  • the size of the detection particles B can be appropriately selected according to the sizes of the well 50 and the through hole 52.
  • the size of the detected particles B can be, for example, a particle size of 50 nm to 80 ⁇ m.
  • the particle size of the detection particles B is, for example, preferably 100 nm to 50 ⁇ m, more preferably 500 nm to 30 ⁇ m, and even more preferably 1 to 20 ⁇ m.
  • the particle size of the detected particles B is preferably 1.5 times or more, more preferably 2 times or more, and 3 times or more the minimum inner diameter of the through hole 52. It is more preferable to have.
  • the number of through holes 52 is not particularly limited and can be appropriately set according to the sizes of the wells 50 and the through holes 52.
  • the number of through holes 52 may be one or more, and may be in the range of, for example, 1 to 100.
  • the number of through holes 52 is preferably two or more from the viewpoint of the outflow efficiency of the liquid in the well 50. Specific examples of the number of through holes 52 include, for example, 2 to 10, 2 to 5, 2, or 3.
  • the position of the through hole 52 is not particularly limited as long as it is the bottom 50a of the well 50. From the viewpoint of facilitating the storage of the screening cells C in the well 50 and the efficiency of the outflow of the liquid in the well 50, it is preferable that one or more through holes 52 are arranged near the center of the bottom 50a.
  • the arrangement of the plurality of through holes 52 is not particularly limited.
  • a plurality of through holes 52 may be arranged together near the center of the bottom portion 50a, or a plurality of through holes 52 may be arranged randomly or in a grid pattern on the bottom portion 50a.
  • the detection particle B is not particularly limited as long as it can capture the target secretion.
  • Examples of the detection particle B include carrier particles on which a substance having binding property to the target secretion is immobilized, cells containing a cell membrane protein having binding property to the target secretion, and the like.
  • the detection particle B is composed of a carrier particle B1 on which a substance having a binding property to a target secretion (hereinafter, also referred to as “binding substance”) B2 is immobilized.
  • binding substance a substance having a binding property to a target secretion
  • the material of the carrier particles B1 is not particularly limited, and those usually used for detecting an antibody or the like can be used.
  • the carrier particle B1 examples include, but are not limited to, beads (magnetic beads, resin beads, etc.), hydrogel particles (sodium alginate gel, agarose gel, etc.), metal particles (gold nanoparticles, etc.) and the like.
  • the binding substance B2 is a substance that specifically binds to the target secretion.
  • the target secretion is an antibody
  • the antigen (antigen peptide, antigen protein, etc.) of the antibody can be used as the target secretion.
  • the target secretion is a cytokine or hormone
  • an antibody that binds to the cytokine or hormone can be used.
  • Immobilization of the binding substance B2 on the carrier particles B1 can be carried out by a known method.
  • the immobilization method include, but are not limited to, a method using a binding pair such as a biotin-avidin bond, and a passive adsorption method.
  • step A a plurality of wells 50 are made to capture the screening cells C and the detected particles B.
  • one screening cell C is stored in one well 50.
  • the operation of capturing the screening cells C in the well 50 (hereinafter, also referred to as “cell capture operation”) and the operation of capturing the detection particles B (hereinafter, also referred to as “detection particle capture operation”) are performed separately. It may be done at the same time.
  • the order of the cell capture operation and the detection particle capture operation is not particularly limited. From the viewpoint that the detection particles B can be easily arranged evenly in the well 50, it is preferable to perform the detection particle capture operation first.
  • the detection particle capture operation involves suspending the detection particles B in an appropriate liquid such as a buffer solution (PBS, physiological saline, etc.) or a medium, and suspending the detection particles B in the suspension.
  • an appropriate liquid such as a buffer solution (PBS, physiological saline, etc.) or a medium
  • suspending the detection particles B in the suspension can be done by supplying a plurality of wells 50. It is not necessary to perform the suspension charging operation for each well 50, and the suspension may be supplied to the opening surfaces of the plurality of wells 50.
  • the detection particles B in the suspension supplied to the wells 50 are stored in each well 50 by their own weight.
  • the detection particles B may be stored in the well 50 while sucking the liquid in the well 50 from a suction hole or the like communicating with the through hole 52.
  • the detection particles B can be stored in the well 50 more quickly.
  • the amount of the detection particles B stored in each well 50 can be adjusted by adjusting the density of the detection particles B in the suspension.
  • the density of the detection particles B in the suspension is not particularly limited as long as the suspension does not impair the fluidity, and can be appropriately selected depending on the size of the detection particles B. Density of particles detected B in the suspension is generally, for example, be a 10 5 to 10 about 12 / mL.
  • the medium for culturing the cells can be appropriately selected according to the type of the screening cells C.
  • the medium When the screening cell C is a mammalian cell, examples of the medium include MEM medium, MEM ⁇ medium, and RPMI medium.
  • the term "appropriate liquid" means a liquid in which screening cells C can survive in the liquid.
  • the suitable liquid is preferably a liquid capable of producing secretions when the screening cell C is a secretion-producing cell. Examples of suitable liquids include buffers and media as described above.
  • the cell capture operation can be performed by suspending the screening cells C in a suitable liquid and supplying the cell suspension to a plurality of wells 50. It is not necessary to perform the injection operation of the screening cell C for each well 50, and the cell suspension is supplied to the surface provided with the openings of the plurality of wells 50 in the member (plate, membrane, etc.) including the plurality of wells 50. do it.
  • the screening cells C in the cell suspension supplied to the wells 50 are stored in each well 50 in units of one cell by their own weight.
  • the storage of the screening cells C in the well 50 may be performed while sucking the liquid in the well 50 from a suction hole or the like communicating with the through hole 52. In this case, the screening cell C can be stored in the well 50 more quickly.
  • the density of the screening cells C in the cell suspension is not particularly limited as long as the cell suspension does not impair the fluidity, and can be appropriately selected depending on the size of the screening cells C. Density Screening cells C in the cell suspension is generally, for example, may be about 10 5 to 10 9 / mL. When a medium is used as the liquid for suspending cells, the medium may be the same as described above.
  • the liquid in the well 50 is discharged from the through hole 52 after the detection particle capture operation, and then the cell suspension containing the screening cell C is contained. May be supplied to the well 50. As a result, the excess medium in the well 50 is removed, so that the efficiency of capturing the screening cells C in the well 50 can be improved.
  • the liquid in the well 50 may be discharged while being sucked from a suction hole or the like communicating with the through hole 52. In this case, the liquid can be efficiently discharged from the through hole 52.
  • the detection particles B and the screening cells C may be suspended in an appropriate liquid, and the suspension may be supplied to a plurality of wells 50.
  • the detection particles B and the screening cells C in the suspension supplied to the wells 50 are stored in each well 50 by their own weight.
  • the detection particles B and the screening cells C may be stored in the well 50 while sucking the liquid in the well 50 from a suction hole or the like communicating with the through hole 52. In this case, the detection particles B and the screening cells C can be stored in the well 50 more quickly.
  • the amount of the detection particles B stored in each well 50 can be adjusted by adjusting the density of the detection particles B in the suspension.
  • the density of the detected particles B in the suspension can be the same as described above.
  • Screening cells C are stored in each well 50 in units of one cell. The density of screening cells C in the cell suspension can be similar to the above.
  • step B screening cells captured in a plurality of wells are allowed to produce secretions.
  • FIG. 2 is a diagram illustrating an example of step B.
  • the screening cell C captured in the well 50 is a secretion-producing cell
  • the screening cell C can be made to secrete the secretion A by incubating the screening cell C in the well 50.
  • Incubation conditions can be appropriately set according to the type of screening cell C. In general, incubation conditions such as 25-38 ° C. and 0.03-5% CO 2 can be used. Specific examples of the incubation conditions include 37 ° C. and 5% CO 2 .
  • Incubation is performed with the screening cells C captured in well 50 present in a liquid such as buffer or medium. Preferably, the inside of the well 50 is filled with a liquid such as a buffer solution or a medium.
  • the secretion A secreted from the screening cell C is the target secretion
  • the secretion A binds to the binding substance B2 and is captured by the detection particle B. Further, a part of the secretion A moves from the through hole 52 to the outside of the well 50 together with the liquid (buffer solution, medium, etc.) in the well 50.
  • the incubation time can be sufficient for the secretion A to be captured by the detection particles B.
  • the incubation time may be appropriately set according to the type of screening cell C, and can be, for example, about 0.5 to 24 hours.
  • step C the target secretion captured by the detection particles is detected.
  • FIG. 3 is a diagram illustrating an example of step C.
  • the target secretion can be detected, for example, by using the detection reagent D.
  • the detection reagent D is composed of, for example, a substance (binding substance) D1 that specifically binds to the target secretion and a labeling substance D2.
  • a substance (binding substance) D1 that specifically binds to the target secretion
  • a labeling substance D2 for example, an antibody that specifically binds to the target secretion can be used.
  • the antibody as the binding substance D1 may be a natural antibody or an unnatural antibody (for example, an antibody fragment such as Fab or scFv). It is preferable that the site of the target substance secretion to which the binding substance D1 binds is different from the site to which the binding substance B2 binds.
  • the binding substance D1 may be an antibody that specifically binds to the constant region of the target secretion antibody.
  • the labeling substance D2 is a substance that generates a signal that can be detected by an arbitrary detection device or the like.
  • the labeling substance D2 is not particularly limited, and a labeling substance generally used in the biochemical field can be used.
  • Examples of the labeling substance D2 include enzyme labeling, fluorescent labeling, radioactive substances and the like.
  • the enzyme label include HRP (Horse Radish Peroxidase), AP (Alkaline Phosphatase) and the like.
  • Fluorescein labels include (FAM (carboxyfluorescein), JOE (6-carboxy-4', 5'-dichloro2', 7'-dimethoxyfluorescein), FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX. Fluorescein such as (5'-hexachloro-fluorescein-CE phosphoroamideite); Cy dye-labeled carboxylic acid such as Cy3, Cy5; AlexaFluor such as Alexa568 and Alexa488; the labeling substance D2 is easy to detect a signal. Therefore, fluorescent labeling is preferable.
  • the detection reagent D can be brought into contact with the target secretion A in the well 50 by dissolving it in an appropriate liquid (buffer solution, medium, etc.) and supplying the detection reagent solution to the well 50.
  • the liquid in the well 50 may be discharged from the through hole 52 before the detection liquid reagent solution is supplied to the well 50.
  • the liquid can be discharged, for example, by removing the liquid from the flow path arranged below the through hole 52.
  • the liquid may be removed from the flow path by suction from a suction hole or the like communicating with the flow path.
  • the detection reagent D supplied into the well 50 comes into contact with the secretion A in the well 50.
  • the detection reagent D binds to the secretion A via the binding substance D1.
  • the detection reagent D is captured by the detection particle B via the secretion A according to the amount of the secretion A captured by the detection particle B. Therefore, by detecting the signal of the labeling substance D2, the secretion A captured by the detection particle B can be detected.
  • the signal of the labeling substance D2 may be detected by a method according to the type of the labeling substance D2.
  • the labeling substance D2 is an enzyme label
  • the color can be developed using the color-developing substrate of the enzyme, and the color-developing signal can be detected using an optical microscope.
  • the labeling substance D2 is a fluorescent label
  • the fluorescent signal can be detected using a fluorescence microscope.
  • the labeling substance D2 is a radioactive substance
  • the radioactive signal can be detected using an X-ray microscope.
  • step D using the detection result in step C as an index, a well in which cells producing the target secretion are captured from a plurality of wells is identified.
  • FIG. 4 is a diagram illustrating an example of step D.
  • the screening cells C, the detection particles B, the secretion A secreted from the screening cells C, and the detection reagent D are present in the plurality of wells 50, respectively.
  • the abundance of the detection reagent D in the well 50 differs depending on the type of the secretion A.
  • Well 50-1 contains screening cells C-1 that secrete secretion A-1.
  • Secretion A-1 is the target secretion and is captured by the detection particle B. Therefore, the detection reagent D is captured by the detection particles B via the target secretion, secretion A-1. As a result, the detection reagent D is accumulated in the well 50-1 according to the amount of the secretion A-1 captured by the detection particles B.
  • Wells 50-2 contain screening cells C-2 that secrete secretion A-2. Secretion A-2 is not the target secretion and is not captured by the detection particle B. Therefore, the detection reagent D flows out from the through hole 52 together with the liquid in the well 50-2 without being captured by the detection particles B.
  • Wells 50-3 contain screening cells C-3 that secrete secretion A-3.
  • Secretion A-3 is not the target secretion and is not captured by the detection particle B. Therefore, the detection reagent D flows out from the through hole 52 together with the liquid in the wells 50-3 without being captured by the detection particles B.
  • the detection reagent D is captured by the detection particles B, the abundance of the detection reagent D in the wells is larger than that in the wells 50-2 and 50-3. .. Therefore, when the signal of the labeling substance D2 is detected, the detected signal intensity is stronger in the well 50-1 than in the wells 50-2 and the well 50-3. Conversely, it can be said that the screening cell C that produces the target secretion is stored in the well having a higher signal intensity of the labeling substance D2 as compared with the other wells. Therefore, a well having a higher signal intensity of the labeling substance D2 as compared with other wells can be identified as a well in which secretory-producing cells producing the target secretion are captured.
  • FIG. 14 (A) shows fluorescence micrographs of wells subjected to steps A to C in Examples.
  • Alexa 488 is used as the labeling substance D2, and it can be confirmed that the fluorescence signal of the well indicated by the arrow is stronger than that of the other wells (FIG. 14 (A) Alexa). Therefore, the well indicated by the arrow can be identified as a well in which secretory-producing cells producing the target secretion are captured.
  • the detection reagent D may be supplied to the well 50 at the same time as the screening cells C and / or the detection particles B.
  • FIG. 5 is a diagram illustrating step A in the case where the detection reagent D is supplied to the well 50 at the same time as the screening cells C and the detection particles B.
  • the detection reagent D When the detection reagent D is supplied to the well 50 at the same time as the screening cells C and the detection particles B, the detection reagent D can be added to the suspension in which the screening cells C and the detection particles B are suspended.
  • the screening cells C and the detection particles B may be suspended in a liquid (buffer solution, medium, etc.) to which the detection reagent D is added. Then, a suspension containing the detection reagent D, the screening cells C, and the detection particles B may be supplied to the well 50.
  • the detection reagent D can be supplied to the well 50 at the same time as either the detection particle capture operation or the cell capture operation.
  • the detection reagent D can be added to an appropriate liquid (buffer solution, medium, etc.) in which the detection particles B are suspended.
  • the detection particles B may be suspended in an appropriate liquid (buffer solution, medium, etc.) to which the detection reagent D is added. Then, the suspension containing the detection reagent D and the detection particles B may be supplied to the well 50.
  • the detection reagent D When the detection reagent D is supplied to the well 50 together with the screening cell C, the detection reagent D can be added to an appropriate liquid (buffer solution, medium, etc.) in which the screening cell C is suspended. Alternatively, the screening cells C may be suspended in a suitable liquid (buffer solution, medium, etc.) to which the detection reagent D is added. Then, the suspension containing the detection reagent D and the screening cell C may be supplied to the well 50. When the operation of discharging the liquid in the well 50 from the through hole 52 is performed between the detection particle capture operation and the cell capture operation, the detection reagent D is added to the suspension of the capture operation performed after the discharge operation. It is preferable to do so.
  • step A the detection reagent D can be supplied to the well 50 at the same time as the cell capture operation and / or the detection particle capture operation, and then step B can be performed in the same manner as described above.
  • the secretion A secreted from the screening cell C is the target secretion
  • the secretion A is captured by the detection particle B via the binding substance B2.
  • the detection reagent D binds to the secretion A via the binding substance D1. Therefore, when the secretion A is the target secretion, the detection reagent D is captured by the detection particles B via the secretion A.
  • step C the signal of the labeling substance D2 may be detected.
  • the signal of the labeling substance D2 can be detected in the same manner as described above.
  • Step D can be performed in the same manner as described above based on the detection result in step C.
  • the detection particle B may be a cell containing a cell membrane protein having a binding property to the target secretion.
  • FIG. 6 shows the state of step C when the detection particle B is a cell containing the cell membrane protein B2'having binding to the target secretion (hereinafter, also referred to as “target cell”).
  • Membrane protein means a protein localized in the cell membrane.
  • the membrane protein may be an integral membrane protein or a peripheral membrane protein, but is preferably an integral membrane protein.
  • the integral membrane protein may be a multiple-penetrating type that penetrates the cell membrane multiple times, or a single-penetrating type that penetrates the cell membrane once.
  • Membrane proteins include GPCRs (G protein-coupled receptors), ligand-gated ion channels, voltage-gated ion channels, transporters and the like.
  • the target cell may be a natural cell or a recombinant cell into which a membrane protein gene has been introduced.
  • the target secretion binds to the region where the membrane protein B2'is exposed on the cell membrane surface.
  • the target secretion is an antibody against a membrane protein specific to a diseased cell (for example, a cancer cell)
  • the diseased cell can be used as a target cell.
  • the target cells are sized to be captured in the well 50 and not pass through the through hole 52.
  • the size of the target cell is, for example, about 1 ⁇ m to 80 ⁇ m.
  • the size of the well 50 can be set based on the total size of the screening cells C and the target cells.
  • the maximum diameter of the circle entering the opening of the well 50 is, for example, larger than the maximum diameter of the target cell and smaller than twice the maximum diameter of the screening cell C.
  • the depth of the well 50 can be set to about 0.5 to 2 times the total value of the maximum diameter of the target cell and the maximum diameter of the screening cell C, and 0.8 to 1.9 times is more. It is preferable, and 0.9 to 1.5 times is more preferable.
  • the size of the well 50 can be set as described above for the detection particle B.
  • the operation of capturing the target cells in the well 50 may be performed at the same time as the cell capture operation, may be performed separately, or may be performed separately. Is preferable.
  • the target cell capture operation is preferably performed before the cell capture operation.
  • the target cell capture operation can be performed by suspending the target cells in an appropriate liquid (buffer solution, medium, etc.) and supplying the target cell suspension to a plurality of wells 50.
  • the target cells C in the target cell suspension supplied to the wells 50 are stored in each well 50 by their own weight.
  • the density of the target cells in the target cell suspension is not particularly limited as long as the target cell suspension does not impair the fluidity, and can be appropriately selected depending on the size of the target cells.
  • the density of the target cells in suspension is generally, for example, may be about 10 5 to 10 9 / mL.
  • Steps B to D can be performed in the same manner as described above.
  • the secretion A secreted from the screening cell C is the target secretion
  • the secretion A binds to the cell membrane protein B2'of the target cell as the detection particle B.
  • the detection reagent D binds to the secretion A. Therefore, the detection reagent D is captured by the cell membrane protein B2'of the target cell via the secretion A. Therefore, based on the signal detection result of the labeling substance D2, the well with high signal intensity can be identified as the well in which the secretion-producing cells producing the target secretion are captured.
  • the screening cells are stored in the wells in units of cells and the secretion assay is performed, the cells do not move during the assay operation. Further, since the target secretion is detected in each well using the detection particles, the distance and time until the target secretion secreted from the screening cells is captured by the detection particles is shortened. Therefore, as compared with the case where the target secretion is detected outside the well, the diffusion of the target secretion is suppressed, and the target secretion can be reliably captured by the detection particles. Therefore, it is possible to accurately identify and obtain the cells that produce the target secretion.
  • the detection reagent not captured by the detection particles flows out from the through hole 52 together with the liquid in the well 50. Therefore, the secretion-producing cells that produce the target secretion can be identified based on the difference in the signal intensity of the labeling substance D2.
  • the screening method of the present embodiment may include other steps in addition to the above steps A to D.
  • steps include, but are not limited to, a washing step (step E), a secretion-producing cell recovery step (step F), and the like.
  • FIG. 7A and 7B are diagrams illustrating an example of a cleaning process.
  • FIG. 7A shows an example in which a cleaning step is performed after step C.
  • FIG. 7B shows the state after the cleaning step.
  • step C when the secretion A secreted from the screening cell C is the target secretion, most of the secretion A is captured by the detection particle B, and the detection reagent D is captured by the detection particle B via the secretion A. Is captured by. However, it is believed that a portion of the detection reagent D remains free (part of which is bound to secretion A) and is present in the well 50. When the secretion A is not the target secretion, all the detection reagents D in the well 50 remain free in the well 50.
  • FIG. 14B shows a fluorescence micrograph of a well in which a washing step was performed after steps A to C in the examples.
  • FIG. 14B it can be confirmed that the fluorescence signals in the wells other than the wells indicated by the arrows have almost disappeared.
  • the wells in which the secretion-producing cells producing the target secretion are captured can be identified more clearly than in FIG. 14 (A).
  • the liquid in the well 50 may be discharged and the liquid may be supplied into the well 50 a plurality of times.
  • the liquid in the well 50 may be discharged and the liquid may be supplied into the well 50 a plurality of times.
  • the cleaning step may be performed after step B.
  • the free secretion A not captured by the detection particles B can be removed from the well 50.
  • the detection reagent D is supplied to the well 50, the amount of the detection reagent D that reacts with the free secretion A is reduced, so that the amount of the detection reagent D used can be reduced.
  • the liquid can be discharged from the through hole 52, for example, by removing the liquid from the flow path arranged under the through hole 52.
  • the well 50 is also maintained in a state of being filled with the liquid.
  • the liquid in the well 50 flows out from the through hole 52 due to gravity, and most of the liquid in the well 50 can be finally removed.
  • the removal of the liquid from the flow path may be performed by sucking the liquid from the suction hole communicating with the flow path.
  • step F Secretory-producing cell recovery step
  • screening cells C are recovered from the wells identified in step D as secretory-producing cells that produce the target secretion.
  • the screening cell C can be recovered by using a known single cell recovery means.
  • the single cell recovery means include a method of recovering cells using a manipulator, a microcapillary, a micropipette, or the like.
  • a second aspect of the present invention is a secretion-producing cell screening kit that screens cells that produce the desired secretion from a plurality of cells.
  • the screening kit comprises a device containing a plurality of wells and carrier particles.
  • the well has a size at which the cells can be captured in units of one cell in a state where one or more of the carrier particles are captured, and has a through hole at the bottom having a size that the cells cannot pass through.
  • the carrier particles have a size that does not pass through the through holes.
  • the screening kit according to the present embodiment can be used for the screening method according to the first aspect.
  • the device includes multiple wells with through holes at the bottom.
  • Examples of the plurality of wells and through-holes include those similar to those mentioned in the above section (Method for screening secretion-producing cells).
  • Examples of the device including a plurality of wells include a device having a configuration as shown in the device 100 of FIG.
  • the device preferably has a flow path that connects to the through hole and is located below the through hole.
  • the flow path arranged below the through hole is preferably a flow path provided along the outer bottom surface of the plurality of wells.
  • the device 1 is provided on the bottom plate portion 2, the cell mounting membrane (cell mounting portion) 6 provided on the bottom plate portion 2 and constituting the cell mounting surface 4, and the cell mounting membrane 6 on the bottom plate portion 2. It has a pair of liquid inflow / outflow portions 8 provided in a partitioned manner, and a flow path 10 provided between the bottom plate portion 2 and the cell mounting membrane 6 and having a flow path end portion 12 extending to the liquid inflow / outflow portion 8. doing.
  • a plurality of wells 50 and through holes 52 leading from the inner bottom surface of the wells 50 to the flow path 10 are formed on the cell mounting surface 4 of the cell mounting membrane 6.
  • a bubble discharge surface 17 that rises in an inclined surface shape as it approaches the suction hole 16A is formed.
  • the bottom plate portion 2 has an elongated rectangular plate shape with a constant wall thickness, and the four corners are chamfered round.
  • a ridge 2B having a constant height is formed on the bottom plate portion 2 along the outer peripheral edge of the bottom surface over the entire circumference.
  • the shape of the bottom plate portion 2 is not limited to the one shown in the drawing, and may be any shape such as a disk shape, an elliptical shape, or a square shape. If a horizontal flow path 10 can be formed, the wall thickness of the bottom plate portion 2 is constant. It does not have to be.
  • the bottom plate portion 2 has a shape that can be placed on the stage of the microscope (for example, a shape similar to a slide glass). It is preferable to have. Generally, it is desirable that the bottom plate portion 2 is made of various plastics that are harmless to cells in terms of molding accuracy and cost, but if necessary, the bottom plate portion 2 is made of any material such as ceramic, glass, or metal. You may be. The surface of the bottom plate portion 2 may be coated with some kind of coating to mitigate the influence on the cells.
  • a rectangular engaging wall 18 having semicircular ends in a plan view so as to surround the central portion stands up vertically from the bottom plate portion 2. It is integrally formed in the state.
  • the engaging wall 18 is not limited to the shape shown in the figure, and may be a simple rectangular shape, a circular shape, an elliptical shape, or the like.
  • the height of the engaging wall 18 in this example is equalized over the entire circumference.
  • An outer frame body 20 is detachably attached to the engaging wall 18 so as to cover the engaging wall 18 from above over the entire circumference.
  • the outer frame body 20 has a rectangular peripheral wall portion 22 having semicircular ends at both ends in a plan view, and a pair of partition portions 28 provided parallel to each other on the inner peripheral side of the peripheral wall portion 22, and the whole is integrated. Is formed
  • the material of the outer frame 20 is not limited, and it is generally desirable that the outer frame 20 is made of various plastics that are harmless to cells in terms of molding accuracy and cost, but if necessary, ceramic, glass, metal, etc. It may be made of any material.
  • a rectangular parallelepiped space is opened between the partition 28 and the partition 28, and the cell mounting membrane 6 is arranged in the space.
  • the upper end of the peripheral wall portion 22 of the outer frame body 20 has a cross-sectional shape that is folded outward over the entire circumference, and inside the folded portion, a narrow engaging groove 24 that opens downward extends over the entire circumference. It is formed at a certain depth.
  • the outer frame body 20 can be attached to and detached from the engaging wall 18 by inserting the upper end portion of the engaging wall 18 into the engaging groove 24 over the entire circumference and elastically tightening the folded portion of the peripheral wall portion 22. It is fixed to.
  • Horizontally projecting protrusions 26 are formed at both tips in the longitudinal direction of the outer frame body 20, and by lifting these protrusions 26 with a fingertip, the engaging wall 18 is pulled out from the engaging groove 24, and the bottom plate portion 2 and the bottom plate portion 2.
  • the outer frame body 20 can be separated.
  • the semicircular region surrounded by the peripheral wall portion 22 of the outer frame body 20 and each partition portion 28 is a liquid inflow / outflow portion 8.
  • a lid portion 14 having a semicircular shape in a plan view and a three-dimensional shape in which the center is raised upward is formed so as to connect the lower end of the peripheral wall portion 22 and the lower end of each partition portion 28.
  • a flow path end portion 12 at both ends of the flow path 10 is formed between the lid portion 14 and the bottom plate portion 2, and the lid portion 14 has a structure that airtightly closes the flow path end portion 12. Since the lid portion 14 is formed in this way, the flow path end portions 12 at both ends of the flow path 10 are sealed, and even when the device 1 is tilted or shaken, the flow path 10 and the flow path are formed. Excessive flow of liquid from side to side through the end 12 is prevented.
  • a circular suction hole 16A is formed at substantially the center of each lid portion 14, and a cylindrical suction port 16 stands upright from the lid portion 14 corresponding to the suction holes 16A. Is formed.
  • the surface of the lid portion 14 around the suction port 16 is used as a liquid storage portion, and the liquid overflowing from the suction port 16 is collected.
  • a recess may be positively formed around the suction port 16 on the upper surface of the lid portion 14.
  • each lid portion 14 On the back side of each lid portion 14, that is, on the ceiling surface of the flow path end portion 12, a foam discharge surface 17 that rises in an inclined surface shape as it approaches the suction hole 16A is formed, and the foam discharge surface 17 has the suction hole 16A at the apex. It has a truncated cone shape.
  • the upper surface of the lid portion 14, that is, the upper surface of the liquid storage portion is also inclined in a truncated cone shape.
  • the liquid storage portion is also formed to be a surface that inclines downward as the distance from the suction port 16 increases, and the liquid spilled from the suction port 16 collects in the peripheral portion of the lid portion 14 away from the suction port 16. It is also possible to reduce the possibility of re-inflow from the suction port 16 and causing contamination.
  • the present invention is not limited to this configuration, and the upper surface of the lid portion 14, that is, the upper surface of the liquid storage portion can be made horizontal by increasing the wall thickness of the lid portion 14 as the distance from the suction port 16 increases.
  • the suction port 16 is erected from the lid portion 14, but instead, the erected suction port 16 may not be formed on the lid portion 14 and the suction hole 16A may be opened as it is.
  • a step portion 34 having a constant height from the lower surface is formed over the entire length of the partition portion 28, and above the step portion 34, the upper end of the partition portion 28 is formed.
  • Each of the two ribs 30 reaching the above is formed so as to extend in the vertical direction.
  • the rib 30 enhances the bending strength of the partition portion 28.
  • the partition portion 28 is formed with an engaging groove 32 having a constant depth that opens on the lower surface of the step portion 34 and an engaging ridge 42 adjacent to the cell mounting membrane 6 side of the engaging groove 32. ing.
  • a square tubular frame 36 having a rectangular shape in a plan view is detachably housed in a square space surrounded by two linearly extending peripheral wall portions 22 and two partition portions 28.
  • a cell mounting membrane 6 is stretched over the entire surface of the lower end of the frame 36 with the wells 50 facing upward, and the lower end of the frame 36 and the cell mounting membrane 6 are joined without gaps over the entire circumference. ing.
  • the frame body 36 is made of a flexible plastic or the like, and when a force to spread it outward is applied, the walls on all four sides expand outward slightly, tension is applied to the cell mounting membrane 6, and the cell mounting film 6 is placed. The slackening of the film 6 can be prevented.
  • the thickness of the cell mounting membrane 6 is not limited, but forms a well 50 capable of capturing screening cells on a cell-by-cell basis, and forms a fine through hole 52 through which a liquid flows from the bottom of the well 50 to the back surface side. From the viewpoint, it is preferably about 5 to 100 ⁇ m, more preferably about 10 to 50 ⁇ m.
  • the cell mounting membrane 6 may be a multilayer membrane having two or more layers. In that case, a through hole to be a well 50 is formed in the upper layer, a through hole to be a through hole 52 is formed in the lower layer, and these two layers are bonded together to form a well 50 and a through hole 52. You may.
  • the material of the cell mounting membrane 6 is not limited, and it is generally desirable to be formed of various plastics that are harmless to cells in terms of molding accuracy and cost, but if necessary, ceramic, polycrystalline or It may be made of any material such as single crystal silicon, an inorganic compound such as glass, and a metal.
  • the well 50 and the through hole 52 can also be formed by etching or photolithography.
  • Examples of the planar shape and size of the well 50 include those exemplified in the above section (Screening method for secretory-producing cells).
  • a plurality of types of frame bodies 36 having cell mounting membranes 6 having different sizes of wells 50 may be prepared and combined with a common bottom plate portion 2 and outer frame body 20 to form the device 1.
  • Examples of the shape and size of the through hole 52 include those exemplified in the above section (Screening method for secretory-producing cells).
  • an engaging groove 44 that opens upward and an engaging ridge 40 that protrudes downward are formed on the outer peripheral surface of the lower end portion of the frame body 36 by forming the shape so as to be folded upward. It is formed over the entire circumference.
  • the depth of the engaging groove 44 and the vertical width of the engaging ridge 40 are substantially constant over the entire circumference of the frame body 36.
  • the engaging ridge 40 is inserted into the engaging groove 32 formed on the lower surface of the outer frame body 20, and the engaging ridge 42 of the outer frame body 20 is inserted into the engaging groove 44 of the frame body 36.
  • the lower end surface of the frame body 36 abuts on the upper surface of the spacer 46 formed on the bottom plate portion 2, and the thickness of the spacer 46 causes the separation amount of the cell mounting membrane 6 from the bottom plate portion 2, that is, the flow.
  • the thickness of the road 10 is accurately defined.
  • a pair of plan-viewing U-shaped spacers 46 are formed inside the engaging wall 18 along the lower end shape of the frame body 36, and the spacers 46 are connected to each other. A notch 47 is formed between them. In a state where the frame body 36 is fixed on the bottom plate portion 2, the liquid flows from the flow path 10 to each flow path end portion 12 through these notches 47.
  • the spacer 46 does not have to have a shape as shown in the drawing, and may come into contact with the lower surface of the frame body 36 at several points. In some cases, the height of the frame body 36 from the bottom plate portion 2 may be accurately defined by engaging with the outer frame body 20 without forming the spacer 46.
  • An inclined surface 38 having a constant width is formed on the inner peripheral surface of the lower end portion of the frame body 36, and the inclined surface 38 protrudes toward the cell mounting membrane 6 as it goes downward. Due to the formation of such an inclined surface 38, the secretory-producing cells are stored when the secretory-producing cells are collected from the well 50 of the cell mounting membrane 6 by an instrument such as a manipulator, a micropipette, or a microcapillary. Even when the formed well 50 is located just before the inner peripheral edge of the frame body 36, the cells can be easily collected. Further, the inclined surface 38 also has an effect of increasing the adhesion area of the cell mounting membrane 6 to the frame body 36 and increasing the bonding strength of the cell mounting membrane 6, and is also useful for increasing the strength of the frame body 36. There is.
  • the bottom plate portion 2, the frame body 36, and the outer frame body 20 are separately formed as shown in FIG. 13, and the frame body 36 is first formed into the outer frame body 20. Then, by fitting the outer frame body 20 into the engaging wall 18 of the bottom plate portion 2, the completed state as shown in FIGS. 8 to 12 is obtained.
  • the engaging ridges 42 of the outer frame body 20 are fitted into the engaging grooves 44 of the frame body 36, and By fitting the engaging ridge 40 of the frame body 36 into the engaging groove 32 of the outer frame body 20, both are firmly fixed by the elasticity of each engaging portion.
  • the inner peripheral surface of the engaging ridge 40 and the outer peripheral surface of the engaging ridge 42 are shaped to be slightly inclined outward as at least one of them goes upward.
  • the flow path end portions 12 located at both ends of the flow path 10 are closed by lid portions 14, and the lid portions 14 are provided with suction ports 16 having suction holes 16A communicating with the flow path 10.
  • the lid portion 14 suppresses the movement of the liquid in the flow path end portion 12 and the flow path 10, and the liquid can be taken in and out of the flow path 10 through the suction port 16. Therefore, even when the device 1 is carried or tilted while the screening cells C are captured in the wells 50 of the device 1 and the flow path 10 is filled with the liquid, the flow path 10 and the flow path end portion 12 are used.
  • the liquid becomes difficult to move, and the so-called sloshing phenomenon can be suppressed. Therefore, it is possible to suppress the problem that a part of the liquid flows into the well 50 through the through hole 52 and the screening cells C and the detection particles B stored in the well 50 are separated.
  • the liquid storage portion (the peripheral portion on the upper surface of the lid portion 14) receives the liquid, and the flow path 10 is again transmitted from the suction port 16. It is possible to suppress the entry into the inside, and it is possible to reduce the risk of contamination from the outside, for example. Further, since the partition portion 28 is formed between the cell mounting surface 4 and the lid portion 14, it is possible to prevent the liquid from flowing to the cell mounting portion 6 even when the liquid is accumulated in the liquid storage portion 14. it can.
  • the cell mounting membrane 6 is positioned at the correct position on the bottom plate portion 2 by bringing the lower end of the frame body 36 into contact with the spacer 46 of the bottom plate portion 2, so that the well 50 and the flow path are formed.
  • the flow of liquid to and from 10 becomes as desired, and highly accurate screening becomes possible.
  • the bottom plate portion 2 and the outer frame body 20 are molded as separate bodies, and the lid portion 14 and the peripheral wall portion 22 are accurately attached to the bottom plate portion 2 simply by attaching the outer frame body 20 to the bottom plate portion 2. Since it can be arranged at any position, the device 1 can be easily assembled. Further, the outer frame body 20 can be removed from the bottom plate portion 2 after use, and maintenance is easy.
  • the cell mounting membrane 6 is simply formed by molding the bottom plate portion 2, the outer frame body 20 and the frame body 36 as separate bodies, and attaching the frame body 36 and the outer frame body 20 to the bottom plate portion 2. ,
  • the lid portion 14 and the peripheral wall portion 22 can be accurately positioned, and the ease of assembly can be improved. Further, the frame body 36 and the outer frame body 20 can be removed from the bottom plate portion 2 after use, and maintenance is easy.
  • the bottom plate portion 2 and the outer frame body 20 are molded as separate bodies, and the outer frame body 20 is attached to the bottom plate portion 2, and the cell mounting portion 6, the lid portion 14, and the peripheral wall portion are simply attached. Since the 22 and the two suction ports 16 can be accurately positioned, it is easy to assemble.
  • the screening cells C, the detection particles B, and the detection reagent D are supplied to the wells 50 from above the cell mounting surface 4 on the cell mounting membrane 6.
  • the liquid in the well 50 can be discharged from the through hole 52.
  • suction from the suction hole 16A discharge can be performed more efficiently.
  • the suction from the suction hole 16A may be performed by sucking the liquid from the suction hole 16A using a micropipette or the like.
  • a suction pump may be connected to the suction port 16 and suction may be performed from the suction hole 16A using the suction pump.
  • the carrier particles are the same as the carrier particles B1 described in the above section (Screening method for secretory-producing cells).
  • the carrier particles may be carrier particles (detection particles B in the first aspect above) on which a substance having a binding property to the target secretion (binding substance) is immobilized.
  • the substance having binding property to the target secretion is the same as the binding substance B2 described in the above section (Screening method for secretion-producing cells).
  • the carrier particles may be those in which the binding substance is not immobilized. In this case, the carrier particles are capable of immobilizing the binding material.
  • the carrier particles may be surface-modified so that the binding substance can be immobilized, or may be formed of a material capable of immobilizing the binding substance.
  • the surface of the carrier particles may be modified on one side of the binding pair. Specific examples include carrier particles coated with streptavidin.
  • the carrier particles may be composed of a material having a property of adsorbing the binding substance.
  • the carrier particles can be polystyrene particles, gold nanoparticles, or the like.
  • the binding substance is not immobilized on the carrier particles, the user can immobilize any binding substance on the carrier particles.
  • the screening kit of the present embodiment may include other configurations in addition to the above device and carrier particles.
  • Other configurations include a reagent for detecting a target secretion, a medium or a buffer solution, a reagent for labeling a binding substance, an instruction manual for use, and the like.
  • the target secretion detection reagent is the same as the detection reagent D described in the above section (Screening method for secretion-producing cells).
  • Examples of the medium or buffer solution are the same as those exemplified in the above section (Method for screening secretion-producing cells).
  • Binding agent labeling reagents are used to modify the binding material so that it can be immobilized on carrier particles.
  • the labeling reagent includes a biotin-labeled reagent.
  • the screening kit of the present embodiment can be used to perform the screening method according to the first aspect, and can be used as described in the above section (Screening method for secretory-producing cells).
  • ⁇ Cell preparation> A gene encoding a mouse anti-mouse RANKL monoclonal antibody (hereinafter referred to as “mouse monoclonal antibody”) was introduced into 293FT cells.
  • the transgenic cells were cultured in MEM ⁇ medium supplemented with fetal bovine serum (FBS) for 48 hours and used as cells for a secretory assay.
  • FBS fetal bovine serum
  • ⁇ Secretory assay> [Step A] Secretory assays were performed using a device with multiple wells with through holes with a pore size of 2 ⁇ m at the bottom. The detection particles prepared in the above ⁇ Preparation of detection particles> were added to the wells pre-wet with phosphate buffer (PBS), and the detection particles were spread in the wells. Subsequently, the cell culture solution prepared in the above ⁇ cell preparation> was seeded in the wells, and the cells were trapped in the wells.
  • PBS phosphate buffer
  • the secreted mouse monoclonal antibody was captured by the goat anti-mouse antibody of the detection particles, and further bound to Alexa488-labeled goat anti-mouse IgG antibody.
  • an antigen-antibody complex consisting of goat anti-mouse antibody-mouse monoclonal antibody-Alexa488-labeled goat anti-mouse IgG antibody was formed.
  • Step C (1) The device after standing in a CO 2 incubator for 3 hours was observed with a fluorescence microscope (model "BZ-9000", manufactured by KEYENCE CORPORATION).
  • FIG. 14 (A) is a typical fluorescence micrograph taken from the bottom side of the well.
  • the nucleus of the cell was detected by the blue fluorescence of Hoechst 33342 (Hoechst; arrow).
  • a mouse monoclonal antibody bound to the detection particles was detected by the green fluorescence of Alexa 488 (Alexa; arrow).
  • Step D (1) From the fluorescent signal detected in step C (1), the well in which the secretion-producing cells secreting the mouse monoclonal antibody were captured could be identified (Merge; arrow).
  • Step E cleaning step
  • the solution in the well was drained through the through hole and PBS was added to the well to wash the detected particles. This removed the unreacted Alexa488-labeled goat anti-mouse IgG antibody.
  • Step C (2) The washed device was observed under a fluorescence microscope.
  • FIG. 14B is a typical fluorescence micrograph taken from the bottom side of the well.
  • the nucleus of the cell was detected by the blue fluorescence of Hoechst 33342 (Hoechst; arrow).
  • a mouse monoclonal antibody bound to the detection particles was detected by the green fluorescence of Alexa 488 (Alexa; arrow).
  • the washing step removed unreacted Alexa488-labeled goat anti-mouse IgG antibody, reducing background. As a result, the fluorescence signal of the mouse monoclonal antibody captured in the detected particles became clear.
  • Step D (2) From the fluorescent signal detected in step C (2), the well in which the secretion-producing cells secreting the mouse monoclonal antibody were captured could be identified (Merge; arrow).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Sustainable Development (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Mycology (AREA)
  • Toxicology (AREA)
  • Clinical Laboratory Science (AREA)
  • Nanotechnology (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Procédé de criblage d'une cellule produisant des sécrétions comprenant le criblage, à partir d'une pluralité de cellules, de cellules qui produisent une sécrétion d'intérêt. Le procédé comprend : une étape A de capture de particules de détection permettant de capturer la cellule et la sécrétion d'intérêt dans une pluralité de puits ayant, dans sa section inférieure, des trous traversants dimensionnés de telle sorte que les cellules ne passent pas à travers ceux-ci; une étape B pour amener les cellules capturées dans la pluralité de puits à produire la sécrétion; une étape C pour détecter la sécrétion d'intérêt capturée par les particules de détection; et une étape D pour identifier, à partir de la pluralité de puits, un puits dans lequel les cellules qui produisent la sécrétion d'intérêt ont été capturées, en utilisant le résultat de détection en tant qu'indice. Le puits a une taille avec laquelle les cellules peuvent être capturées dans des unités d'une cellule tout en capturant une ou plusieurs des particules de détection.
PCT/JP2020/036534 2019-09-30 2020-09-28 Procédé de criblage de cellules produisant des sécrétions et kit de criblage de cellules produisant des sécrétions WO2021065766A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP20871602.7A EP4039789A4 (fr) 2019-09-30 2020-09-28 Procédé de criblage de cellules produisant des sécrétions et kit de criblage de cellules produisant des sécrétions
US17/754,049 US20220381768A1 (en) 2019-09-30 2020-09-28 Method for screening secretion-producing cells and kit for screening secretion-producing cells
JP2021551221A JPWO2021065766A1 (fr) 2019-09-30 2020-09-28
KR1020227010340A KR20220070220A (ko) 2019-09-30 2020-09-28 분비물 산생 세포의 스크리닝 방법, 및 분비물 산생 세포의 스크리닝 키트
CN202080067740.0A CN114450594A (zh) 2019-09-30 2020-09-28 分泌物生产细胞的筛选方法、及分泌物生产细胞的筛选试剂盒

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2019178577 2019-09-30
JP2019-178577 2019-09-30

Publications (1)

Publication Number Publication Date
WO2021065766A1 true WO2021065766A1 (fr) 2021-04-08

Family

ID=75338243

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/036534 WO2021065766A1 (fr) 2019-09-30 2020-09-28 Procédé de criblage de cellules produisant des sécrétions et kit de criblage de cellules produisant des sécrétions

Country Status (7)

Country Link
US (1) US20220381768A1 (fr)
EP (1) EP4039789A4 (fr)
JP (1) JPWO2021065766A1 (fr)
KR (1) KR20220070220A (fr)
CN (1) CN114450594A (fr)
TW (1) TW202130803A (fr)
WO (1) WO2021065766A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009034047A (ja) * 2007-08-02 2009-02-19 Toyama Prefecture 細胞のスクリーニング方法
JP4603894B2 (ja) 2002-12-03 2010-12-22 ユセベ ファルマ ソシエテ アノニム 抗体産生細胞を同定するためのアッセイ
WO2017057234A1 (fr) * 2015-09-29 2017-04-06 東京応化工業株式会社 Substrat, structure, procédé de fabrication de structure, procédé de tri de cellules, procédé de fabrication de cellules, et procédé de production de sécrétions
KR20180039005A (ko) * 2016-10-07 2018-04-17 울산과학기술원 나노 입자 검출 장치 및 이를 이용한 나노 입자 검출 방법
JP2019178577A (ja) 2018-03-30 2019-10-17 株式会社Ｌｉｘｉｌ 小便器

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013012452A2 (fr) * 2011-03-03 2013-01-24 The Regents Of The University Of California Appareil à nanopipettes pour la manipulation de cellules
EP3529611B1 (fr) * 2016-10-23 2022-11-23 Berkeley Lights, Inc. Procédés de criblage de lymphocytes b
JP2021505157A (ja) * 2017-12-07 2021-02-18 マサチューセッツ インスティテュート オブ テクノロジー 単一細胞分析
CN115698254A (zh) * 2020-05-25 2023-02-03 东京应化工业株式会社 靶标粒子的分离方法及系统

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4603894B2 (ja) 2002-12-03 2010-12-22 ユセベ ファルマ ソシエテ アノニム 抗体産生細胞を同定するためのアッセイ
JP2009034047A (ja) * 2007-08-02 2009-02-19 Toyama Prefecture 細胞のスクリーニング方法
WO2017057234A1 (fr) * 2015-09-29 2017-04-06 東京応化工業株式会社 Substrat, structure, procédé de fabrication de structure, procédé de tri de cellules, procédé de fabrication de cellules, et procédé de production de sécrétions
KR20180039005A (ko) * 2016-10-07 2018-04-17 울산과학기술원 나노 입자 검출 장치 및 이를 이용한 나노 입자 검출 방법
JP2019178577A (ja) 2018-03-30 2019-10-17 株式会社Ｌｉｘｉｌ 小便器

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Single Domain Antibodies", METHODS IN MOLECULAR BIOLOGY, vol. 911, 2012
SKRLEC KSTRUKELJ BBERLEC A: "Non-immunoglobulin scaffolds: a focus on their targets", TRENDS BIOTECHNOL, 27 April 2015 (2015-04-27)

Also Published As

Publication number Publication date
EP4039789A1 (fr) 2022-08-10
CN114450594A (zh) 2022-05-06
EP4039789A4 (fr) 2023-10-04
TW202130803A (zh) 2021-08-16
US20220381768A1 (en) 2022-12-01
KR20220070220A (ko) 2022-05-30
JPWO2021065766A1 (fr) 2021-04-08

Similar Documents

Publication Publication Date Title
JP6825065B2 (ja) 分泌物の多細胞アッセイにおけるマイクロ流体デバイスおよびその使用方法
US11359175B2 (en) Substrate, structure, structure-manufacturing method, cell-sorting method, cell-manufacturing method, and secretion-producing method
JP7416749B2 (ja) 細胞分泌物の分析のための装置及び方法
EA025219B1 (ru) Микролуночный чип для скрининга целевых клеток и его применение
US7374906B2 (en) Biological assays using gradients formed in microfluidic systems
AU2003287170A1 (en) Biological assays using gradients formed in microfluidic systems
WO2021065766A1 (fr) Procédé de criblage de cellules produisant des sécrétions et kit de criblage de cellules produisant des sécrétions
CN112210487A (zh) 高通量筛选分泌抗原特异性抗体浆细胞的装置及方法
Ekert et al. Integration of the Immune System Into Complex In Vitro Models for Preclinical Drug Development
Ekert et al. 14 Integration of the
CN115097145A (zh) 一种基于荧光标记免疫法的蛋白质定量检测方法及其使用的微流控芯片

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20871602

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021551221

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020871602

Country of ref document: EP

Effective date: 20220502